Download the regulation of the differential expression of the human globin

J . Cell Sci. Suppl. 4, 319-336 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
319
THE REGULATION OF THE DIFFERENTIAL
EXPRESSION OF THE HUMAN GLOBIN GENES
DURING DEVELOPMENT
D. J. W EATH ERALL
MRC M olecular Haematology Unit, Nuffield Department o f Clinical Medicine,
University o f Oxford, John Radcliffe Hospital, Oxford, UK
INTRODUCTION
Although a great deal is known about the structure and molecular pathology of the
human haemoglobin genes it is still not clear how their differential expression during
normal development is regulated. As well as being of considerable interest to
developmental geneticists, this problem has important practical implications. Varia­
bility in the expression of the foetal globin genes plays a major role in modifying the
clinical course of some of the common genetic disorders of adult haemoglobin
production. If it were possible to prevent the switching off of foetal haemoglobin
production after the neonatal period, or to reactivate it even partially, we would have
an extremely valuable approach to the management of these conditions, which are
globally the commonest single gene disorders.
Here I shall summarize what has been learnt from the experimental systems that
are being used to study the regulation of the developmental changes in globin gene
expression. It will be possible to touch on only those areas that seem to be of
particular promise for future work. Several recent reviews cover human haemoglobin
genetics and the developmental biology of haemoglobin in more detail (Wood &
Weatherall, 1983; Collins & Weissman, 1984; Orkin & Kazazian, 1984; Weatherall,
Higgs, Wood & Clegg, 1984; Weatherall & Wainscoat, 1985); original references to
much of the experimental work described here will be found in these articles.
THE ORGANIZATION OF THE HUMAN GLOBIN GENES
The structures of the different haemoglobins that are synthesized during embry­
onic, foetal and adult life are summarized in Fig. 1. They are all tetramers consisting
of two pairs of unlike peptide chains, each associated with a haem molecule. Normal
adults have a major haemoglobin, haemoglobin A ( ocifii), and a minor component
called haemoglobin A2 (# 2^ 2)- The main haemoglobin in foetal life is haemoglobin F ,
which has a chains combined with y chains ((XzY?). It is a mixture of two different
molecular forms that differ only by one amino acid in their y chains, glycine or
alanine at position 136; the y chains that make up these two types of foetal
haemoglobin are thus referred to as Gy and Ay. In embryonic life there is yet another
D. jf. W eatherall
320
series of haemoglobins in which the a chains are replaced by g chains and the y and
/3 chains by £ chains.
As shown in Fig. 1 the globin genes are organized in two families, an a-like gene
cluster on chromosome 16 and a /3-like cluster on chromosome 11. W ithin each
complex the genes, together with several inactive pseudogenes, are all in the same 5'
to 3' orientation and are arranged in the order in which they are expressed at different
stages of development. However, comparison with other vertebrate species suggests
that it is unlikely that there is any general relationship between gene order and
developmental expression.
T h e /3-like genes are distributed over approximately 60 kb (103 bases) and are
arranged in the order 5 ' - £ - Gy—Ay—Xffi—d —f i - 3 '. T h e a-like genes form a smaller
cluster on chromosome 16, in the order S ' - g - ^ g - ' ^ a - a l - a l - ' i ' . T h e t[>/J, tpfand
^ar genes are pseudogenes. T h e position of the introns is shown in Fig. 1. T h e 5'
flanking regions of each of the genes contain two regions of homology. One, the A T A
box, is 20—3 0 base-pairs (bp) upstream from the RNA initiation site; the other, the
C C A A T box, is 70—90 bp upstream from this site. Each or gene is located within a
region of homology approximately 4 kb long, interrupted by two small nonhomologous regions. T h e exons and the first introns of the two a globin genes have
identical sequences. T h e two g genes are also highly homologous. Like the a l and a2
genes, the Gy and Ay genes appear to be virtually identical, reflecting a process of
gene matching during evolution. In fact, the Gy and Ay genes on one chromosome
are identical in the region 5' to the centre of the large intron, yet show greater
divergence in a 3' direction. At the boundary between the conserved and divergent
regions there is a block of simple sequence, which may be a ‘hotspot’ for the initiation
of recombination events that lead to unidirectional gene conversion.
Several classes of repetitive sequences have been identified in the eyd/3 globin gene
cluster. T h ere are single Alu repeat sequences upstream from the y globin genes and
1 kb
i---------------
Ç2 e 2
Hb Gower 1
’ 2 '2
Hb Portland
E m bryo
<*2 §2
Hb Gower 2
HbA2
Foetus
Fig. 1. T h e genetic control of human haemoglobin.
A dult
Differential expression of human globin genes
321
from the (3 genes, and inverted pairs of Alu sequences upstream from the £ and
6 genes and downstream from the /? globin gene. The three inverted pairs are
orientated tail to tail with about 800 bp of non-repetitive DN A between them. The
second major class of repeat sequences belongs to the K pn family. One copy lies
downstream from the ¡3 globin gene; another between the e and y genes. The latter
region, over 6 kb in length, has been sequenced and at the end near the y globin
gene has strong homology with the retrovirus long-terminal repeat (see Collins &
Weissman, 1984).
Table 1. The globin gene switches during normal human development
g—> a\ and a2
e —»Gy and Ay
Gy and Ay —* 5 and /?
Foetal Gy/Ay—»adult Gy/Ay
GLOBIN GENE EXPRESSION DURING HUMAN DEVELOPMENT (T a b le 1)
The embryonic haemoglobins are synthesized mainly during the period when
erythropoiesis is confined to the yolk sac. Throughout the rest of foetal life the liver
and spleen are the main sources of red cell production, although the marrow starts to
produce red cells during the second trimester and becomes the major erythropoietic
site during later foetal life.
Recently, the patterns of globin chain production at very early stages of embryonic
development, during the transition from yolk sac (primitive) to hepatic (definitive)
erythropoiesis, have been analysed (Peschle et al. 1984, 1985). During the 4th to 5th
week fan d £ chains and very small quantities of y chains are synthesized. During the
6th to 7th week a, g, e, Gy and Ay chains are produced by the remaining primitive
erythroblasts, and a , £, Gy and Ay chains by the definitive line. By the 7th to 8th
week e and g chain synthesis is no longer detectable and the main globin chains
synthesized are a, Gy and Ay; ¡3 chain production is just detectable at this time and
gradually increases, so that at about 10 weeks it constitutes about 10% of total non­
a chain production. Thus there appears to be a slight asynchrony in the switch from
g to or compared with £ to y chain production; the g—* a chain transition is completed
slightly earlier.
From the 10th to the 33rd week of gestation the main globin chains produced are
a > GY, AY and (3. Assessment of the output of the two linked or globin genes by
m RNA analysis suggests that they are expressed in the ratio a 2 / a l of 1-5—3-0/1
throughout foetal life (Liebhaber & K an, 1981; Orkin & Goff, 1981); this does not
change during development and is the same as that observed in normal adults. The
relative rates of Gy and Ay chain production are also constant throughout foetal life at
a Gy /Ay ratio of approximately 3 /1 (Nute, Pataryas & Stamatoyannopoulos, 1973).
Between the 32nd and 36th week of gestation the relative rate of ¡3 chain synthesis
322
D. J . Weatherall
increases and that of y chain production declines, so that at birth /3 chain synthesis
constitutes approximately 5 0 % of non-ar chain synthesis. After birth the level of
y chain production declines steadily and that of ¡3 chain production increases ; at the
end of the first year y chain synthesis reaches the low level characteristic of adult life.
During the first few months of life the Gy / Ay ratio changes from 3 /1 to 2 /3
(Schroeder e ta l. 1972). Delta chain production has been observed as early as 32
weeks gestation; ô chain activation lags behind that of /? chains, and the adult
/3 /<5 chain synthesis ratio is only reached at about 4 - 6 months after birth.
Although there has been extensive debate about the intercellular distribution of
different haemoglobins during development it is now believed that the transition
from embryonic to foetal and foetal to adult haemoglobin production occurs within
the same erythrocyte populations. This conclusion is also consistent with recent
studies of the patterns of y and /? chain production in red cell colonies grown from
foetal and neonatal blood. It is also clear that the type of globin chains produced at
different stages of development is not related to the site of erythropoiesis ; both fan d
s chains are synthesized in both primitive and definitive cell lines (Peschle et al.
1984, 1985) and the switchover from y to /? chain production occurs synchronously
throughout the liver, spleen and bone marrow during the later stages of foetal
development (Wood & Weatherall, 1973; Wood et al. 1979). Furthermore, the
transition from y to ¡3 chain synthesis is related closely to gestational age and not to
birth ; premature infants continue to synthesize relatively high levels of y chains until
about 40 weeks gestation (Bard, 1975).
Thus the various developmental haemoglobin transitions occur within the same
cell populations, are synchronized between the changing sites of erythropoiesis
during development, and are closely related to the gestational age of the foetus.
These changes in haemoglobin constitution are associated with other developmental
modifications of the red cell, particularly the switching on of one of the carbonic
anhydrase isozymes and several alterations in surface antigens.
FOETAL HAEMOGLOBIN PRODUCTION IN NORMAL ADULTS
Normal adults produce small amounts of haemoglobin F , which range from 0 -3 to
0 -8 % of the total haemoglobin. Analysis of the intercellular distribution suggests
that this is confined to a small population of adult red cells, which for this reason are
called F cells, although they also include large amounts of haemoglobin A. The
relative proportion of F cells is remarkably constant in different individuals and
appears to be genetically determined, though how many genes are involved is not
clear. T he relative number of F cells increases during rapid regeneration of the
marrow after periods of transient aplasia. Studies of disorders such as polycythaemia
vera or chronic myeloid leukaemia suggest that F cells are not clonally derived but
arise from the same stem cell pool as adult haemoglobin-containing red cells; the
apparent restriction of y chain production to a small proportion of adult red cells may
be an artefact of the methods used to assess the intercellular distribution of foetal and
adult haemoglobin (see Wood & Weatherall, 1983).
Differential expression of human globin genes
323
THE REGULATION OF GLOBIN GENE EXPRESSION DURING DEVELOPMENT
Because so little is known about the developmental regulation of gene expression,
and the lack of good experimental models with which to investigate this problem, our
current approaches to the analysis of gene switching are, of necessity, indirect. Areas
of study that are providing some information on this question are summarized in
Table 2.
Table 2. Methods used to analyse the regulation o f globin genes during developm ent
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Méthylation state and DNase I sensitivity of the globin genes
Mutations associated with persistent y chain synthesis
Haemopoietic-cell transplantation across developmental stages
Gene expression in colony systems in vitro
Gene expression in neoplastic cell lines
Transgenic mice
Manipulation of gene switching in vivo
Changes in globin gene structure during development
Several aspects of the structure of the globin gene complexes have been studied in
an attempt to understand the mechanism of the differential expression of their
constituent loci during development. Comparisons of the primary sequences of the
individual genes and their flanking regions have shown that, in general, they rapidly
lose homology upstream and downstream of the transcribed sequences except in the
case of those that have diverged recently. However, interspecies comparisons have
not identified any sequences either 5' or 3' to the structural genes that might be
candidates for regulation of their differential expression during development. I shall
return to some very recent studies of globin gene mutations that are relevant to
this question in a later section. There is no evidence that there are any major
rearrangements of the globin gene clusters at different stages of human development.
The globin genes are hypomethylated in tissues in which they are expressed and
are differentially methylated at different stages of development. It appears that at all
stages of ontogeny the /3-like globin genes show a strong correlation between their
methylation state and expression. Similarly, there is a strong tissue and agedependent relationship between their differential sensitivity to nuclease digestion.
DNase I hypersensitive sites have been found 5' to the Gy, Ay, 6 and /? genes in foetal
liver haemopoietic tissue but only 5' to the 8 and /3 genes in adult haemopoietic
tissues. These changes are presumably due to alterations in chromatin structure,
both around the cluster reflecting its potential expression in erythroid cells, and
within a cluster as each gene is activated at different times during development
(Collins & Weissman, 1984).
5-Azacytidine, a cytidine analogue that is incorporated into DNA but cannot be
methylated, appears to be able to activate y gene expression in adult animals and in
humans (DeSimone, Heller, Hall & Zwiers, 1982; Ley et al. 1982). Demethylation
of the y chain genes was observed in erythroid cells after azacytidine treatment of
324
D .J . Weatherall
humans and experimental animals, although this was also true of the e genes in the
case of humans, yet the latter were not expressed. These studies also suggest that
hypomethylation may be necessary for expression. I shall return to this question
later.
Table 3. M utations associated with persistent haemoglobin F production
(1) Sickle cell anaemia and /3 thalassaemia
(2) Hereditary persistence of foetal haemoglobin (HPFH)
Deletion
Non-deletion
linked to j8 globin gene cluster
unlinked to ¡3 globin gene cluster
M utations associated with persistent y chain synthesis in adu lt life
T h e mutations that are characterized by persistent foetal haemoglobin production
are summarized in Table 3. The most important are the ¡3 thalassaemias and ¡3 chain
haemoglobin disorders such as sickle cell anaemia, and a family of conditions that are
characterized by persistent foetal haemoglobin synthesis without any major haema­
tological abnormalities, hereditary persistence of foetal haemoglobin (H P F H ).
Gene-analysis studies of H P FH have shown that the condition can be divided into
deletion and non-deletion forms. More recently it has been found that the latter
group can be subdivided into conditions in which the genetic determinants are linked
to the ¡3 globin gene cluster and those in which they segregate independently from
the cluster.
Sickle cell anaem ia an d ¡3 thalassaemia. The factors that are involved in the
production of elevated levels of foetal haemoglobin in the blood of individuals with
these conditions are extremely complex. Haemoglobin F protects against sickling. In
¡3 thalassaemia, cells that produce y chains are at an advantage since the latter
combine with excess a chains; red cell precursor destruction in this disorder results
from the deleterious effect of excess or chains that accumulate due to defective
¡3 chain synthesis. Thus in both of these disorders red cell precursors or mature red
cells that have the capacity for producing y chains undergo intense selection, either
in the marrow or in the peripheral blood. On the other hand, it is equally clear that
genetic factors are also involved in haemoglobin F production in these conditions. In
some individuals with sickle cell anaemia or (3 thalassaemia, in whom unusually high
levels of haemoglobin F afford protection from the effects of the disease, it is poss­
ible to find normal or heterozygous family members with increased levels of
haemoglobin F . Thus it appears that a gene (or genes) for heterocellular H P FH (see
below) is segregating in these families. This is not always the case, however.
Another approach to this problem has been developed recently. Scattered
throughout the ¡3 globin gene cluster there are a number of restriction fragment
length polymorphisms (R F L P s ), which can be used as genetic markers for following
Differential expression of human globin genes
325
mutations of the ¡3 globin genes (Antonarakis, Boehm, Giardina & Kazazian, 1982).
It has been found that particular arrangements of these R F L P s (haplotypes) may be
associated with an unusually high production of haemoglobin F in individuals with
sickle cell anaemia or /3 thalassaemia (Wainscoat et al. 1985a). This suggests that
there may be a genetic determinant within or linked to the /3 globin gene cluster,
which, since these haplotypes are not associated with increased haemoglobin F
production in symptomless heterozygotes, results in an unusually high level of
y chain production in states of increased erythropoiesis. The only clue to the nature
of this determinant is the recent observation that an alteration in the relative amount
of Gy to Ay chain production in individuals with sickle cell anaemia, and possibly an
increased capacity for y chain synthesis, may be associated with a single base change,
C —>T, at position —158 in the Gy globin gene (Gilman & Huisman, 1984). I shall
return to the significance of this finding in a later section.
The <5/3 thalassaem ias an d deletion forms of HPFH. These conditions are all
characterized by long deletions of the y<5/3 globin gene cluster. Their rather daunting
nomenclature is explained in the legend to Fig. 2. They constitute a spectrum of
disorders in which absent ¡3 chain production is compensated by persistent y chain
synthesis. If compensation is more or less complete the condition is haematologically
normal and is called H P F H ; if there is less efficient y chain synthesis, and hence
unbalanced globin chain production, the condition is called <5/3 thalassaemia.
However, in all these disorders there is an absolute increase in y chain production in
adult life that cannot be accounted for by cell selection. This suggests that the
deletions that cause them must be responsible for the high output of y chains.
T he different deletions that produce 8/3 thalassaemia and H P FH are summarized
in Fig. 2. A question of major interest is whether a comparison of their site and size
can explain the difference in phenotype between H PFH and 6/3 thalassaemia and
hence tell us anything about the position of putative regulatory regions in the
y<5)3 gene cluster. A comparison of particular interest is the 5' extent of the deletions
that cause either (6/3)° thalassaemia or (<5/3)° H P FH in Black populations (Fig. 2).
These deletions end within lkb of each other in the Alu repeat region 5' to the
8 globin gene. The deletion that causes H P FH ends in the middle of the upstream
Alu repeat while that which causes 8/3 thalassaemia ends 1 kb downstream from the
latter in the other Alu repeat (see Collins & Weissman, 1984). Is this region involved
in the regulation of y and /3 chain synthesis during development? While this may be
the case, the fact is that both these deletions cause considerably elevated levels of
y chain production in adult life; the difference between them is only a matter of
degree. Furthermore, a Greek family has recently been described in which
homozygotes have the clinical picture of a mild form of ¡3 thalassaemia and
heterozygotes have either normal or marginally elevated haemoglobin F levels, and
yet this condition results from a deletion that removes the entire region occupied by
the Alu repeat sequences (Wainscoat et al. 19856).
Another interpretation of these different phenotypes is that they do not depend
directly on the region of DNA that is deleted but rather on the particular sequences
that are brought into apposition to the j3 globin gene complex by the deletion.
326
D. J . W eatherall
O 2 4 6 8 10 kb
i- 1- 1 1 1 <
>3 Ok b
>2 5 k b
!h
Hh
P° THAL. ( USA )
P° THAL.( Indian)
P°THAL.( Dutch)
Hb LEPORE
Ï THAL.
(S P )0 HPFH(USA)
(S P )0 HPFH ( Ghana]
Hb KENYA HPFH
( S P)°THAL.( Sicily )
(S P) °THAL.( Spanish
(SP)°THAL.( Indian)
(5 p ) °THAL. ( Greek )
vmm'M
.INVERTED
vmv//Æ
(AySP)°THAL.(Turki
(AX5P)°THAL( India
( A jj¿ p)° THAL.( Chint
( AjfS P)°THAL.( Malay
p )°( Anglo-saxo
(e Jfáp)°( Dutch)
( i yá p)° ( Mexican )
(£¡r¿P)°( Scot /Irish
( ¿ Y Í
■ j
“W J
Wi
1
mzzzm.
Fig. 2. T h e deletions that give rise to different forms of (5/3 thalassaemia and hereditary
persistence of foetal haemoglobin (H P F H ). (5/3)°, (Ay<5/3)° etc. indicate that these
conditions are characterized by an absence of 8 and /3, or Ay, <5 and ft chain production.
T H A L means that persistent y chain production is insufficient to com pensate for lack of
/? chains and hence that the condition has a thalassaemic phenotype. T h e country or
nationality in which the lesion was first described is added to define the particular
m utation. References to original descriptions are given by Collins & Weissman (1984) and
W eatherall & W ainscoat (1985).
Perhaps, it has been argued, in some forms of H PFH sequences are brought in from
3 ' to the ¡3 globin gene complex that act as cis enhancers, thus allowing expression of
the foetal genes in adult life (Collins & Weissman, 1984). However, as shown in
Fig . 2, the 3' ends of all these deletions are different; do they all contain rather
similar enhancer sequences? T h is seems unlikely, and perhaps the most attractive
hypothesis to explain the phenotypic variability of these deletions is that the
y d ¡3 globin gene cluster is organized into two chromatin domains, one surrounding
the foetal genes, the other flanking the <5 and ¡3 genes, both with distinct 5' and 3'
borders. Interference with any of these domain boundaries may prevent activation of
the adult domain and leave the foetal genes unrepressed. T h is hypothesis was
discussed in detail by Bernards & Flavell (1980). Additional deletion mutations of
the y d ¡3 globin gene cluster are being mapped in an attempt to clarify these issues.
However, because they all cause such a major disruption of the gene complex, their
D ifferential expression o f human globin genes
327
-CCAAT
— ATA
-300
-200
- I ---------------------------- ih
r
Gy
-100
f
-202 OG :-158 O f ;
-300
'
-200
'(t-196 G*T
-198 T-C
-100
t
'
-117 G-A
Fig. 3. T h e non-deletion forms of H P F H due to lesions involving the Gy or Ay genes.
study may be of limited value for providing information about gene control during
normal development.
N on-deletion HPFH. Because these conditions are not associated with major
disruptions of the ¡3 globin gene cluster they are of much greater potential interest for
analysing the regulation of gene expression during development. As mentioned
earlier, the genetic determinants for some of these conditions map within or near the
13 gene complex while others segregate independently from the ¡3 globin gene
markers and hence must be at a considerable distance away on chromosome 11 or
even on another chromosome.
Some well-defined forms of non-deletion H PFH in which the genetic determinants
are within the f3 globin gene cluster are summarized in Fig. 3. In the Gy (3+ variety of
H P F H , which has been found exclusively in Black populations, heterozygotes
produce approximately 2 0 % haemoglobin F of the Gy type and there is ¡3 chain
production in cis. Sequence analyses of the Ay gene and of the Alu repeat region 5' to
the <5 gene have shown no abnormality (Jones, Goodbourn, Old & Weatherall, 1985;
D r Oliver Sm ithies, personal communication). However, a single point mutation
(C-^-G) has been identified 202 base-pairs 5' to the CAP site of the Gy gene (Collins
et al. 1984). A related disorder, called Greek H P F H , in which heterozygotes pro­
duce approximately 15 % foetal haemoglobin in adult life has also been analysed at
the molecular level. T h e sequence of the Ay globin genes showed no abnormalities
except for a single change, G ^ A at position —117, i.e. 117 bases 5' to the CAP site
(Collins et al. 1985; Gelinas et al. 1985). T h is change has been found in two
unrelated heterozygotes. In a similar disorder observed in Italy a single base change,
G —>T, has been found at position —196 in the Ay gene (Giglioni et al. 1984). T h is
has also been observed in a Chinese individual with a similar phenotype (D r G.
Stamatoyannopoulos, personal communication).
In the British form of non-deletion H PFH (Weatherall et al. 1975a) homozygotes
have about 2 0 % haem oglobinF, which is mainly of the Ay variety; heterozygotes
have between 3 and 10% haemoglobin F . We have studied several heterozygotes for
this condition at birth and followed their pattern of haemoglobin F production
328
D. J . Weathera.il
during the first year of life (Wood et al. 1982). The Gy /Ay chain production ratio is
normal at birth but the rate of decline of haemoglobin F production is retarded and
the adult pattern of predominantly Ay chain synthesis appears during the first few
months of life. These observations suggest that the primary defect in this condition
affects a regulatory region involved in the neonatal suppression of Ay chain pro­
duction; when the Gy and Ay loci are fully activated in foetal life the expression of the
genes is normal. Recently, we have sequenced the Gyand Ay genes from an individual
homozygous for this condition; both genes are normal except for a T —>C change at
position —198 in the Aygene (Tate e t al. unpublished data).
These interesting new forms of H P F H are summarized in Fig. 3. They appear to
result from a series of single base changes clustered 5' to the Gy or Aygenes, upstream
from the C C A T and A TA boxes. In view of the developmental history of foetal
haemoglobin production in the British variety, it is possible that these mutations
alter D N A /protein interactions that are involved in the neonatal suppression of y
gene synthesis.
Finally, there are non-deletion HPFH-like conditions associated with relatively
low levels of haemoglobin F production in heterozygotes. Although some of them
may be caused by determinants that map within the ¡3 globin gene cluster (Old et al.
1982) several families have now been reported in which this is not the case (Gianni
et al. 1983). This is the first evidence for the existence of genes that influence globin
chain synthesis that are not linked to the globin gene cluster. Currently, linkage
studies are being carried out to attempt to determine the chromosomal location of
these putative regulatory regions. Recently, we have established a linkage for a form
of H P F H of this type to a restriction fragment length polymorphism defined by a
mini-satellite probe (Jeffreys, Wilson & Thein, 1985).
H aemopoietic cell transplantation between developm ental stages
T he pattern of switching between foetal and adult haemoglobin is similar in sheep
and man, and thus it has been possible to perform interdevelopmental stage haemo­
poietic cell transfer studies in this species (Wood et al. 1985). The rationale for these
experiments was as follows. Foetal haemopoietic cells, obtained from liver and bone
marrow, can be transplanted into irradiated lambs in which the switch from foetal to
adult haemoglobin synthesis is already complete. If the transplanted cells switch over
to adult haemoglobin synthesis immediately, it would imply that switching is
determined mainly by the microenvironment of the erythroid progenitors in the bone
marrow. If, on the other hand, switching occurs in the donor cells at about the same
time as it would had the cells remained in the foetus, this would point to the existence
of an intrinsic regulatory mechanism or ‘developmental clock’ within the foetal
haemopoietic stem cells. Finally, if the transplanted cells continue to produce foetal
haemoglobin indefinitely, it suggests that gene switching is under the control of
a regulatory mechanism that is only present at a particular time during foetal
development and hence had been bypassed by the transplantation.
Given the technical difficulty of these studies, the results of a large number of
experiments are now reasonably consistent (Wood et al. 1985). Foetal haemopoietic
Differential expression of human globin genes
329
cells transplanted into newborn animals continue to synthesize foetal haemoglobin
and then gradually switch over to adult haemoglobin production. The timing of the
transition is related to the gestational age of the foetus from which the donor cells
were obtained, although it may be accelerated very slightly in the recipient. In the
converse experiment, adult bone marrow cells transplanted into a foetus synthesize
predominantly adult haemoglobin, implying that once the switch has occurred it
is irreversible. T o date, the results of these transplant experiments are compatible
with a ‘developmental clock mechanism’ for the regulation of foetal globin gene
expression.
Gene expression in neoplastic cell lines
The notion that analysis of gene expression in haematological neoplasms might
provide some useful models for studying the developmental genetics of haemoglobin
is not new. It has been known for some time that infants with juvenile chronic
myeloid leukaemia (JC M L ) revert to a pattern of red cell protein production that is
very similar to that observed late in foetal life (Weatherall, Edwards & Donohoe,
1968; Weatherall et al. 19756). Their haemoglobin consists predominantly of Hb F
with a marked reduction of Hb A2 and carbonic anhydrase, another protein that is
switched on during the later part of foetal development. Unfortunately it has not
been possible to establish JC M L cells in culture.
There are, however, several established cell lines that are of potential interest for
studying the developmental genetics of haemoglobin. The main stimulus to these
studies came from the observation that a mouse erythroleukaemic cell line (M E L ),
first established by Charlotte Friend, when induced by dimethyl sulphoxide, haem
or other agents, undergoes terminal erythroid differentiation and synthesizes
haemoglobin, in this case of the adult variety (Marks & Rifkind, 1978). T h e human
cell line K 562 was originally derived from a patient with transforming chronic
granulocytic leukaemia (C G L ) (Lozzio & Lozzio, 1975). Although there are slight
variations between different K 562 lines, most of them synthesize predominantly £
and g chains with smaller amounts of a and y chains when induced with haem or
other agents (Rutherford, Clegg & Weatherall, 1979). No ¡3 chain production has
been found in these cells. The ¡3 globin genes are intact, have a normal structure
and are expressed normally when cloned and transferred to COS cells. Curiously,
of the two or genes, only a l is expressed in K 562 cells (D. R. Higgs, unpublished
observation).
Another line, in this case derived from a patient with erythroleukaemia and called
Human Erythroleukaemia Line (H E L ), after induction, produces mainly Ay and
Gy chains with some or but no (3 chains (Martin & Papayannopoulou, 1982). Another
human leukaemia cell population that synthesized only haemoglobin F was also
derived from an adult patient with transforming C G L , but a permanent line could
not be established (Potter et al. 1984).
Thus it appears that some adult-derived leukaemia cell lines can be induced to
express their embryonic and, or, foetal globin genes. There does not appear to be any
structural abnormality of the later-developmental globin genes, which are not
330
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expressed in these cells; in a sense they appear to be ‘frozen’ at an early develop­
mental stage, similar to JC M L cells described earlier. Hence they offer a useful
model for chromosome or gene transfer experiments for defining the possible role of
trans- acting regulatory factors that might be involved in the expression of globin
genes during development.
In ta ct chromosome or gene transfer experiments
A number of experiments have been carried out that have asked whether there is
any evidence for developmental-stage-specific trans regulation of the eyd/3 globin
gene complex. The interpretation of these studies is based on the assumption that
some of the mouse or human malignant cell lines that can be induced to produce
adult or foetal haemoglobin, described above, are ‘fixed’ at specific developmental
stages. It has been found that if chromosome 11, cosmids containing the human ¡3, y
and e genes, or plasmids containing the ¡3 genes alone, are inserted into mouse
erythroleukaemia (M E L ) cells there is a significant increase in ¡3 globin gene ex­
pression after induction of haemoglobin synthesis, whereas there is no increase in
the expression of the y or e genes (Willing, Nienhuis & Anderson, 1979; Wright,
De Boer, Grosveld & Flavell, 1983). Similarly, when intact human chromosomes 16
are transferred into M E L cells there is expression of the or globin genes but not
of the embryonic g globin genes (Zeitlin & Weatherall, 1984). Furthermore, if
chromosome 16 of K 562 cells, in which the embryonic globin genes are active, is
transferred into M E L cells, no g gene expression is observed (Anagnou et al. 1985).
Recently, chromosome 11 derived from the human cell line H E L has been
transferred into M E L cells (Papayannopoulou et al. 1985). As mentioned earlier,
H E L cells synthesize embryonic and foetal non-or chains but do not produce adult
f3 globin chains. After transfer, the ¡3 globin genes were activated, suggesting that
they are transcriptionally competent and thus that they may be responding to a
positive trans- acting element within the M E L environment. Presumably they fail to
express in the H E L environment because of the absence of this factor or the presence
of a iraws-acting inhibitor of ¡3 globin gene expression.
T h e results of these experiments suggest that, if it is assumed that M E L cells are
‘adult’ in character, they lack the appropriate stage-specific developmental trans
regulatory factors that are required for the expression of the embryonic or foetal
globin genes but produce trans- acting factors capable of supporting a and /3 gene
expression. However, other data have not all been consistent with this interpretation.
T h e or globin genes, when part of chromosome 16, show induction, as does the
f3 globin gene on chromosome 11, but when a globin genes are introduced into M E L
cells as part of a cosmid or plasmid they are expressed at a high level, independent of
induction. This behaviour contrasts with that of the ¡3 globin genes, which still show
induction dependence when introduced in either a cosmid or plasmid. Nevertheless,
this is a promising approach to the definition of trans regulators and it should be
possible to expand these studies provided that a source of embryonic or foetal
recipient cells can be obtained for similar transfer experiments.
Differential expression of human globin genes
331
Gene expression in colony system s in vitro
There is an extensive literature on the differential expression of the y and ¡3 globin
genes in clonal colonies derived from B F U -E s and C F U -E s , erythroid progenitor
cells which can be defined by their size and time of appearance in culture in vitro.
This work is summarized in the published accounts of three recent conferences on
gene switching (Stamatoyannopoulos & Nienhuis, 1979, 1981, 1983).
The expression of the foetal and adult globin genes in colonies reflects the stage of
maturation of the individuals from which the progenitors were obtained. Colonies
derived from foetuses produce predominantly y chains, whereas those obtained from
adults synthesize mainly (3 chains although there is always a higher proportion of y
chains produced in adult-derived colonies than is present in adult red cells. Both y
and ¡3 chain genes are expressed in the same colonies and there is a continuum in the
relative proportion of y and /8 chain production in B F U -E s obtained from newborns
at different gestational ages. The latter argues against there being specific stem cell
populations that are programmed for foetal or adult globin chain synthesis.
A variety of modifications of experimental conditions, particularly those that affect
the growth of colonies, can change the relative expression of y and ¡3 globin genes in
erythroid colonies. This may be because the relative expression of y and ¡3 genes is
related to the maturity of the progenitors. At least in some species there appears to be
asynchrony of y and ¡3 gene expression during colony maturation, with y genes
expressed earlier than ¡3 genes. Recent experiments in which the absoute amount of y
and ¡3 chain accumulation in colonies has been estimated suggest that this may not
always be the case, particularly in human B F U -E s . However, when absolute levels
of haemoglobin accumulated per cell are calculated, the amount in vitro is always
considerably below that obtained in vivo. Thus the attention that has been paid
to the higher proportion of haemoglobin F in adult B F U -E s has not yet been
demonstrated to have any bearing on globin gene regulation in vivo.
Perhaps the most interesting observation relating to the regulation of gene
switching that has arisen from studies of erythroid colonies is that the expression of
the y genes in both adult B F U -E s and those from individuals with various genetic
conditions associated with persistent y chain production, including deletion H P F H ,
can be ‘switched off’ by a factor that is present in foetal sheep sera. This has led to the
development of a model in which switching occurs when the appropriate receptors
for this putative inhibitory factor are expressed during the later stages of foetal
development.
Several other models of the regulation of the y and ¡3 globin genes have been
derived from analyses of the pattern of globin gene expression in colonies. For
example, it has been suggested that programming may reflect a ‘decision’ by early
progenitors to move to terminal differentiation in which y chain production is more
likely, rather than to go through further divisions and differentiation steps that make
/3 gene expression more probable. This stochastic model of differentiation has been
extended to encompass the regulation of foetal and adult globin gene expression
during in vivo development. However, the in vitro colony model has not provided
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D .J . Weatherall
any clear insights into how these different pathways of differentiation might be
mediated or regulated.
In vivo modification o f yglobin gene expression
As mentioned earlier, the administration of agents such as 5-azacytidine, which
cause hypomethylation of genes, is associated with a modest increase in y chain
production in patients with sickle cell anaemia or /? thalassaemia (Ley et al. 1982;
Charache e t al. 1983). Since this effect can be obtained with cytotoxic agents that do
not cause hypomethylation of D N A it has been suggested that the effect on y chain
production results from perturbations of the patterns of erythroid maturation
(Letvin et al. 1985). It is possible that both mechanisms play a role.
CONCLUSIONS
Clearly, it is impossible to synthesize the diverse information outlined in this
review and provide a coherent model for the regulation of globin gene expression
during normal human development. One of the great difficulties in this field is
uncertainty about whether the mutations that are associated with persistent y chain
production, or for that matter the experimental models that have been used to study
the differential expression of the foetal and adult globin genes, have any real
relevance to the normal globin gene switching mechanism. They may have, but only
with respect to a limited part of what must be an extremely complex multi-step
regulatory system.
T h e consistent changes in chromatin and the methylation state of the /J globin gene
cluster that are associated with activation of the different gene loci at various stages of
development provide an anatomical explanation for the activity of these loci but
tell us nothing about how these changes are mediated. However, the gene or
chromosome transfer experiments suggest that there may be developmental-stagespecific trans factors involved in the regulation of these genes, presumably by
interacting with chromatin. This is a very promising area for further work although,
since all these experiments rely on the expression of genes in neoplastic cell lines, the
results have to be interpreted with particular caution. Equally interesting is the
possibility that the ‘upstream’ mutations, which have been found recently in some
varieties of non-deletion H P F H , will provide a clue as to the site of interactions
between chromatin and regulatory proteins. Thus at least we have some indications
of what might be the most productive area of investigation for trying to characterize
the mechanisms of regulation at the chromosomal level. Similarly, recent successes
with the expression of ‘foreign’ globin genes in transgenic mice point the way to how
we might learn more about the tissue specificity of globin gene expression (Chada
et al. 1985).
This may be as far as we can go in the immediate future. The central question
remains, however. How is the differential expression of the globin genes during
development actually timed? All we know at the moment is that it is related fairly
closely to gestational age and that it is not tissue dependent. The only experimental
Differential expression of human globin genes
333
data relating to this question, derived from the sheep transplant model, suggest that
there may be a ‘developmental clock’ built into the haemopoietic stem cell. Here we
have a serious conceptual difficulty because there is no obvious model with which to
analyse time-related events; none of the forms of H PFH is, strictly speaking, a
heterochronic mutation. That is, these conditions are not characterized by a change
in the time of globin gene switching; in the only form of non-deletion H P FH that has
been studied during the period of switching the timing of the transition from foetal to
adult haemoglobin production was completely normal; there was a delay in the rate
o f decline of foetal haemoglobin production suggesting that the mutation involved
the mechanism of adult suppression of y chain production (Wood et al. 1982; Tate
et al. unpublished data).
One of the main difficulties in designing experiments to ask questions about the
timing of events during development is the lack of any clear concept of how the
process might be mediated. One possibility, that should be amenable to investi­
gation, is that differential globin gene expression is related to the number of haemo­
poietic stem cell divisions. A clock based on such a mechanism is feasible; in the
mouse it has been estimated that there may only be a limited number of haemopoietic
stem cell divisions during foetal development. However, our preliminary experi­
ments in sheep suggest that the foetal haemopoietic stem cell is extremely resistant to
perturbation by agents such as busulphan, and that chronic hypertransfusion of the
foetus, which might be expected to reduce the number of stem cell divisions, has very
little effect on the timing of the transition from foetal to adult haemoglobin (W . G.
Wood & C. Bunch, unpublished observations).
Clearly, our current understanding of the developmental genetics of haemoglobin
is at an extremely rudimentary stage. However, it is apparent that there are several
promising areas for further work and that the globin gene model may still have
something to offer to developmental biology.
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